Polyene macrolide antibiotics such as amphotericin B are important in the treatment of systemic fungal infections. Amphotericin B forms sterol dependent ion channels in fungal cell membranes, which lead to loss of intracellular components and cell death. The ion channels are formed by spontaneous assembly of several amphotericin B and membrane sterol molecules. Antifungal selectivity is determined by the greater susceptibility of ergosterol containing fungal membranes compared with cholesterol containing mammalian membranes. We will explore the structural requirements for ion channel assembly and sterol recognition by preparing structural analogs of polyene macrolides. Roflamycoin, an ion channel forming polyene macrolide, is our initial synthetic target. We have developed a very concise approach to this natural product that will be suitable for the rapid assembly of structural analogs. We will prepare four stereoisomers of roflamycoin to determine the importance of stereochemistry on ion channel formation. Roflamycoin and other polyene macrolides are rod shaped molecules that have a polar, polyol surface and a non-polar polyene surface. The ion channels are held together by intramolecular hydrogen bonds between the polar hydroxyl groups of each macrolide molecule. The hydroxyl groups may need to be aligned in a precise manner to hold the ion channel together, or their alignment may be much less critical because extensive hydration will stitch together any stereochemical arrangement. Preparing stereochemical analogs of roflamycoin and assaying their ability to form ion channels will allow us to determine the importance of polyol stereochemistry on ion channel assembly and stability. We will prepare several analogs of roflamycoin where the polyene has been replaced by another rigid, hydrophobic backbone. The polyenes give these macrolides their rod-like shapes and are essential for ion channel activity. The polyene is also the contact surface between the macrolide and the sterol. It is expected to play a dominant role in sterol recognition, but almost nothing is known about this function. We will use planer and non-planer aromatic backbones to replace the polyene. Studying their ion channel forming ability and sterol dependence will clarify the role of the polyene in sterol recognition and ion channel formation.